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.

Description:
RELATED APPLICATIONS  
       [0001]    This application claims priority to GB1223309.4 filed Dec. 21, 2012 which is expressly incorporated by reference herein in its entirety. 
         [0002]    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  
       [0003]    The broad spectrum antimicrobial agent, 5-chloro-2-(2,4-dichlorophenoxy)phenol (also referred to as “triclosan” or “Irgasan®”, which is Ciba Specialty Chemical&#39;s brand name for triclosan), has been commonly used since the early 1970&#39;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. 
         [0004]    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. U.S. Pat. No. 5,447,849 to Toora teaches the use of a combination of cefsulodin and triclosan for selective growth of  Yersinia enterocolitica.  U.S. Pat. No. 5,741,663 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 et al. teaches the addition of triclosan in the range of 100 nM to 10 μM to culture medium (which was also supplemented with ampicillin) for distinguishing genetically modified (transformed)  E. coli  showing vector-mediated expression of the gene for enoyl-ACP reductase (fab1). 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 
       [0005]    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. 
         [0006]    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. 
         [0007]    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. 
         [0008]    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. 
         [0009]    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 substitution. 
         [0010]    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. 
         [0011]    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. 
         [0012]    In preferred embodiments the selective agent is a composition for inclusion in a bacterial growth medium. 
         [0013]    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 O-linkage. The triclosan is bonded to the anomeric carbon of a carbohydrate moiety. 
         [0014]    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. 
         [0015]    In preferred embodiments the triclosan glycoside derivative is selected from:
       triclosan-α-D-arabinopyranoside,   triclosan-β-D-arabinopyranoside,   triclosan-α-D-galactopyranoside,   triclosan-β-D-galactopyranoside,   triclosan-α-D-glucopyranoside,   triclosan-β-D-glucopyranoside, and   triclosan-α-D-mannopyranoside.       
 
         [0023]    In preferred embodiments, the selective agent has the general formula (I): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0024]    wherein R 1  is a glycone (i.e. R 1  is a sugar moiety). Preferably the R 1  group includes a pyranose ring. 
         [0025]    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. 
         [0026]    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%. 
         [0027]    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. coli  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. 
         [0028]    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. 
         [0029]    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. 
         [0030]    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-β-D-thiogalactopyranoside, p-nitrophenyl-α-L-arabinopyranoside or p-nitrophenyl-β-D-xylopyranoside. 
         [0031]    The selective agent is suitably provided in the medium at a suitable concentration which allows target cells to grow, whilst inhibiting non-target cells. 
         [0032]    Suitably the invention also provides a medium, as defined above, in contact with a mixed population of target and non-target cells. 
         [0033]    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. coli.  The target cells are  Salmonella  spp and the non-target cells are coliforms, preferably  E. coli.  In this embodiment the selective agent is preferably triclosan β-D-galactopyranoside. 
         [0034]    In alternative embodiments a culture medium for enumeration of  Campylobacter  spp 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 β-D-galactopyranoside or triclosan α-D-arabinopyranoside. The invention also provides use of a selective agent or medium as defined above for the growth and enumeration of  Campylobacter.    
         [0035]    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. 
         [0036]    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.       
 
         [0039]    The method may be performed without contacting the sample with a pre-enrichment medium that lacks the selective agent. 
         [0040]    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. 
         [0041]    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. 
         [0042]    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 instructions for performing the method according to the invention. 
         [0043]    According to the present invention there is also provided 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:
       triclosan-α-D-arabinopyranoside,   triclosan-β-D-arabinopyranoside,   triclosan-α-D-galactopyranoside,   triclosan-β-D-galactopyranoside,   triclosan-α-D-glucopyranoside,   triclosan-β-D-glucopyranoside, and   triclosan-α-D-mannopyranoside.       
 
         [0051]    The terms arabinoside, galactoside, glucoside and mannoside are used herein as shorthand to refer to the relevant glycone moiety. 
         [0052]    In preferred embodiments, the selective agent has the general formula (I): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0053]    wherein R 1  is a glycone (i.e. R 1  is a sugar moiety). Preferably the R 1  group includes a pyranose ring. 
     
    
     
       DESCRIPTION OF FIGURES 
         [0054]    Preferred embodiments of the present invention will now be more particularly described by way of example only with reference to the accompanying drawings, wherein: 
           [0055]      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-β-D-galactoside (2 (μg/ml) at 37° C. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0056]    Triclosan Derivatives 
         [0057]    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, α-D-arabinopyranoside, β-D-arabinopyranoside, α-D-galactopyranoside, β-D-galactopyranoside, α-D-glucopyranoside, β-D-glucopyranoside, and α-D-mannopyranoside. 
         [0058]    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-β-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 (μg/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. 
         [0059]    All of the organisms that were tested were inhibited by triclosan but several were totally resistant to its glycosides (Table 1). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 MICs of Triclosan-glycosides in Nutrient Broth No. 2 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 α-D-Glucoside 
                 α-D-Galactoside 
                 α-D-Arabinoside 
                 α-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 
                 &gt;256 
                 &gt;256 
                 128 
                 256 
                 &gt;256 
                 &gt;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 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;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 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
               
               
                 
                   marcescens 
                 
               
               
                 OCC 217 
               
               
                   
               
             
          
           
               
                   
                 β-D-Glucoside 
                 β-D-Galactoside 
                 β-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 
                 &gt;256 
                 256 
                 256 
                 256 
                 &gt;256 
                 &gt;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 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;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 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
               
               
                 
                   marcescens 
                 
               
               
                 OCC 217 
               
               
                   
               
             
          
           
               
                   
                 α-D-Glucoside 
                 α-D-Galactoside 
                 α-D-Arabinoside 
                 α-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 
                 &gt;256 
                 &gt;256 
                 128 
                 128 
                 256 
                 256 
               
               
                 
                   cereus ATCC 
                 
               
               
                 14579 
               
               
                 
                   Bacillus 
                 
                 1 
                 1 
                 32 
                 32 
                 256 
                 &gt;256 
                 32 
                 32 
                 128 
                 256 
               
               
                   subtilis  NCTC 
               
               
                 10073 
               
               
                 
                   Enterococcus 
                 
                 1 
                 8 
                 128 
                 128 
                 &gt;256 
                 &gt;256 
                 128 
                 128 
                 256 
                 256 
               
               
                 
                   faecalis 
                 
               
               
                 ATCC 29212 
               
               
                 
                   Enterococcus 
                 
                 1 
                 4 
                 256 
                 256 
                 &gt;256 
                 &gt;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 
                 &gt;256 
                 &gt;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 
                 &gt;256 
                 &gt;256 
                 128 
                 128 
                 128 
                 128 
               
               
                   viridans  OCC 
               
               
                 234 
               
               
                   
               
             
          
           
               
                   
                 β-D- 
                   
                 β-D- 
                   
                 β-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 
                 &gt;256 
                 256 
                 &gt;256 
                 &gt;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 
                 &gt;256 
                 128 
                 128 
               
               
                   
                 OCC 234 
               
               
                   
                   
               
             
          
         
       
     
       EXAMPLE 1  
       [0060]    Acetylation of Sugars 
         [0061]    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 (2×50 ml), saturated aqueous NaHCO 3  solution (2×50 ml) and brine (2×50 ml). The dichloromethane layer was then dried with magnesium sulphate, filtered and concentrated in vacuo to yield the product as a white powder. 
         [0000]    
       
                 
         
             
             
         
       
     
       EXAMPLE 2  
       [0062]    Bromination 
         [0063]    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 2 Cl 2  (2×100 ml). The combined extracts were washed with saturated aqueous NaHCO 3  solution (2×100 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. 
         [0000]    
       
                 
         
             
             
         
       
     
       EXAMPLE 3  
       [0064]    Method 1 for Glycosidation 
         [0065]    Use of the Koenigs-Knorr method ensured that only the trans anomeric form of the glycoside was formed. 
         [0066]    Triclosan (4.04 g, 14.0 mmoles) was dissolved in 100 ml of water containing 14 ml of a 1M sodium hydroxide solution (14mmoles) 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. 
         [0000]    
       
                 
         
             
             
         
       
     
       EXAMPLE 4 
       [0067]    Method 2 for Glycosidation 
         [0068]    As an alternative method, both α- and β-anomers are formed and may be separated using flash chromatography. 
         [0069]    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 (2×150 ml) and brine (2×150 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 hexane/ethyl acetate). 
         [0000]    
       
                 
         
             
             
         
       
     
         [0070]    General Procedure: Deprotections 
         [0071]    Under argon, the protected sugar (1 eq) was dissolved in anhydrous MeOH (1 ml per mmol). K 2 CO 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  
       [0072]      FIG. 1  shows the growth of a mixed culture of  E. coli  607 and  Salmonella typhimurium  722 in Nutrient Broth No. 2 containing triclosan-β-D-galactoside (2 (μg/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 1.0×10 4  to 1.86×10 4  after 30 minutes, numbers then decreased to 3.98×10 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 2.51×10 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  
       [0073]    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.    
         [0074]      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-β-D-galactoside and triclosan-α-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. baumanii  strains tested (Table 2). Triclosan-β-galactoside was particularly active in inhibiting two carbapenemase-producing strains of  Klebsiella pneumoniae  when used at 5 μg/ml while all  Campylobacter  strains were resistant to triclosan-α-arabinoside at concentrations as high as 50 μg/ml. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 MIC values for  Campylobacter  and interfering organisms 
               
               
                 (μg/ml) 
               
             
          
           
               
                   
                 Triclosan-α- 
                 Triclosan-β- 
               
               
                 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 
                 &gt;50 
                 &gt;50 
               
               
                   Campylobacter jejuni  OCC1596 
                 &gt;50 
                 &gt;50 
               
               
                   Campylobacter jejuni  OCC1261 
                 &gt;50 
                 &gt;50 
               
               
                   Campylobacter jejuni  OCC2340 
                 &gt;50 
                 &gt;50 
               
               
                   Campylobacter lari  OCC1598 
                 &gt;50 
                 &gt;50 
               
               
                   Campylobacter coli  OCC2774 
                 &gt;50 
                 &gt;50 
               
               
                   Klebsiella pneumoniae  KPC2 
                 &gt;50 
                 5 
               
               
                   Klebsiella pneumoniae  7KPC3 
                 &gt;50 
                 5 
               
               
                   Enterobacter cloacae  CTXm9 
                 10 
                 5 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 7  
       [0075]    Various glycoside derivatives of triclosan were added to 64 μg/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-β-D-thiogalactopyranoside, p-nitrophenyl-α-L-arabinopyranoside and p-nitrophenyl-β-D-xylopyranoside. Concentration of inducer in all cases was 100 μg/ml. 
         [0076]    From Table 1 it can be seen that the addition of triclosan-α-D-mannoside to Nutrient Broth at 64 μg/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-β-D-arabinoside at 16 μg/ml would allow the selective recovery of  Salmonella  and triclosan-α-D-glucoside would allow the selective recovery of pathogenic strains of  Streptococcus.    
         [0077]    It was also observed that the addition of inducers substantially increased the inhibitory activity of the triclosan glycosides. 
       EXAMPLE 8  
       [0078]    Method for the Determination of Minimum Inhibitory Concentrations in Nutrient Agar 
         [0079]    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. 
         [0080]    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 μg/ml to 0 μg/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 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. 
         [0081]    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. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 MICs of Triclosan-glycopyranosides (μg/ml) in Nutrient Agar containing 
               
               
                 0.1 μg/ml of inducer 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                   
                   
                 β-D- 
               
               
                   
                 No. of 
                 α-D- 
                 α-D- 
                 α-D-gluco- 
                 α-D- 
                 β-D- 
                 β-D- 
                 gluco- 
               
               
                 Organisms 
                 Strains 
                 arabinopyranoside 
                 galactopyranoside 
                 pyranoside 
                 mannopyranoside 
                 arabinopyranoside 
                 galactopyranoside 
                 pyranoside 
               
               
                   
               
             
          
           
               
                 
                   Bacillus cereus 
                 
                 1 
                 128 
                 &gt;256 
                 64 
                 128 
                 256 
                 256 
                 256 
               
               
                 
                   Bacillus subtilis 
                 
                 1 
                 128 
                 128 
                 16 
                 256 
                 256 
                 64 
                 256 
               
               
                 
                   Ent. faecalis 
                 
                 1 
                 256 
                 &gt;256 
                 128 
                 256 
                 256 
                 &gt;256 
                 &gt;256 
               
               
                 
                   Ent. faecium 
                 
                 1 
                 256 
                 &gt;256 
                 128 
                 256 
                 256 
                 &gt;256 
                 &gt;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 
                 &gt;256 
                 128 
                 128 
                 128 
                 256 
                 &gt;256 
               
               
                 
                   Str. pneumoniae 
                 
                 1 
                 64 
                 256 
                 32 
                 64 
                 64 
                 64 
                 128 
               
               
                 
                   Str. pyogenes 
                 
                 1 
                 128 
                 &gt;256 
                 64 
                 64 
                 64 
                 256 
                 64 
               
               
                 
                   Str. viridans 
                 
                 1 
                 128 
                 &gt;256 
                 64 
                 128 
                 128 
                 256 
                 256 
               
               
                 
                   Aer. hydrophila 
                 
                 1 
                 128 
                 &gt;256 
                 128 
                 256 
                 256 
                 256 
                 &gt;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 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;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 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256 
                 &gt;256