Method for detection of microorganisms

Abstract

A method is for the detection of presence or absence, quantification, and identification of target microorganisms, such as bacteria, bacterial fragments (e.g., LPS, endotoxin), viruses, fungi as well as other pathogens by means of chemiluminescence. The method include providing a medium with one or more target analytes, target microorganisms or target metabolites, adding a dioxetane compound to the medium so that the dioxetane compound emits light, and detecting the emitted light.

What is claimed is: 
     
         1 . A method for the detection of a target microorganism, comprising:
 a) providing a medium comprising one or more target analytes, target microorganisms or target metabolites;   b) adding a dioxetane compound to the medium so that the dioxetane compound emits light; and   c) detecting the emitted light, 
 wherein said dioxetane compound has the Formula I: 
       
         
           
           
               
               
           
         
       
       wherein
 R 1  is an analyte-responsive group selected from the group consisting of an enzyme-labile group and a boron-containing group having the formula —B(Z)(Z′) or -B(Z″) 3   - Kat + ; 
 Z and Z′ are independently selected from the group consisting of R 4  and OR 5 , 
 wherein R 4  is selected from the group consisting of —OH, -O -  Kat+, C1-C4 alkyl, C2-C4 heteroalkyl, C2-C4 alkenyl, C2-C4 heteroalkenyl, C2-C4 alkynyl, C2-C4 heteroalkynyl, C5-C6 aryl, C5-C6 heteroaryl, C6-C10 aralkyl, and C6-C10 heteroaralkyl, 
 and R 5  is selected from the group consisting of —H, C1-C4 alkyl, C2-C4 heteroalkyl, C2-C4 alkenyl, C2-C4 heteroalkenyl, C2-C4 alkynyl, C2-C4 heteroalkynyl, C5-C6 aryl, C5-C6 heteroaryl, C6-C10 aralkyl, and C6-C10 heteroaralkyl, or 
 wherein two R 4 , two R 5  or one R 4  and one R 5  together with their intervening atoms form a 5- to 7-membered heterocyclic ring; 
 Z″ is selected from the group consisting of F, Cl, Br, and I; 
 Kat +  is an organic or inorganic cation; 
 L is a self-immolative linker selected from the group consisting of 
                     
                     
                     
                     
                     
 and 
                     
 which, upon acting of an analyte on the analyte-responsive group R 1 , is released from the remainder part of the compound of Formula I, wherein X is connected to R 1  and is either absent or selected from the group selected from —O—, —NH—, and —N + (R G ) 2 —, provided that when R 1  is —B(Z)(Z′) or —NO 2 , X is absent; X′ is selected from the group consisting of S, O, NH, and NR G ; and L is optionally functionalized with a peptide, an endolysine or a protein; provided that:
 when R 1  is an enzyme-labile group, n is 1 and m is 1 or n is 0 and m is 1; 
 when R 1  is the enzyme labile group 
                     
 or 
                     
 n is 0 and m is 1; and 
 when R 1  is —B(Z)(Z′) or -B(Z″) 3   - Kat + , n and m are both 0 or both 1; 
 R A  and R C  are independently selected from H and R 2 —Q—, provided that R A  and R C  are not both H; 
 R B  is H; 
 R 2  is a group selected from the group consisting of cyano, nitro, aryl, alkenyl, 
                     
 carbonyl having the structure 
                     
 and amide having the structure 
                     
 wherein Y is H, a C1-C12 alkyl, or an alkali metal ion, 
 and Y′ and Y″ each independently is selected from the group consisting of H and C1-C12 alkyl, or together with the nitrogen atom form a heterocyclic structure; 
 
 R 3  is H, F, Cl, Br, I, CF 3 , or R 2 —Q—; 
 Q is group comprising a pi-system that is conjugated with the pi-system of the central aromatic ring of the compound of Formula I; 
 R D  is selected from the group consisting of a linear or branched C1-C18 alkyl and C3-C7 cycloalkyl; 
 R E  and R F  each independently is selected from the group consisting of a branched C3-C18 alkyl and C3-C7 cycloalkyl, or R E  and R F  together with the carbon atom to which they are attached form a fused, spiro or polycyclic ring; and 
 R G  each independently is selected from C1-C12 alkyl. 
 
     
     
         2 . The method of  claim 1 , wherein
 (i) Z″ is F; or   (ii) Kat +  is an alkali metal cation; or   (iii) L is                         or   (iv) X is —O— or —N+(CH 3 ) 2 —; or   (v) Y′ and Y″ together with the nitrogen atom form a maleimide group.   
     
     
         3 . The method of  claim 1 , wherein said dioxetane compound is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       and 
       
         
           
           
               
               
           
         
       
       . 
     
     
         4 . The method of  claim 3 , wherein said dioxetane compound is selected from the compounds of Formula II, IIa III IIIa, IIIb VII, VIIa, VIIb, VIII, and VIIIa: 
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       wherein Y is H, an optionally substituted C1-C12 alkyl, or an alkali metal ion. 
     
     
         5 . The method of  claim 1 , wherein said target microorganism is selected from a bacteria, yeast, mold, and virus. 
     
     
         6 . The method of  claim 5 , wherein said bacteria is selected from the group consisting of a  Salmonella ;  Listeria ;  Staphylococcus ;  E. coli ;  carbapenem-resistant bacteria ;  Campylobacter ;  Bacillus ;  Clostridium ;  Mycobacterium ;;  Streptococcus ;  Pseudomonas ;  Enterococcus ;  Citrobacter ;  Coliform ;  Cronobacter sakazakii ;  Geobacillus stearothermophilus ;  Vibrio ;  Legionella pneumophilia ; apyrase-containing bacterium; extended-spectrum beta-lactamase (ESBL)-producing enterobacterium; and  Prevotella ; said yeast is a  Candida ; and said virus is a virus of the Caliciviridae family. 
     
     
         7 . The method of  claim 6 , wherein said Salmonella is  S. enterica ; said  Listeria  is  L. monocytogenes ; said  Staphylococcus  is S. aureus or a methicillin-resistant  Staphylococcus aureus  (MRSA); said  carbapenem-resistant bacteria  is  Pseudomonas aeruginosa  or  Klebsiella pneumonia ; said  Campylobacter  is  C. jejuni ,  C. coli , or  C. lari ; said  Clostridium is C. perfringens  or  C. difficile ; said  Mycobacterium  is  M. tuberculosis ; said  Streptococcus  is  S. agalactiae  or  S. pyogenes ; said  Pseudomonas  is P. aeruginosa ; said  Enterococcus  is vancomycin-resistant  Enterococcus  (VRE); said apyrase-containing bacterium is  Shigella ; said  Candida  is  Candida albicans ; said virus of the Caliciviridae family is a Lagovirus, Norovirus, Sapovirus, Nebovirus, or Recovirus. 
     
     
         8 . The method of  claim 1 , wherein
 (i) the method is for the detection of bacterial endotoxins via detection of limulus factor C, in which said compound of the Formula I, wherein R 1  is Boc-Val-Pro-Argininyl or Boc-Asp(OBzl)-Pro-Argininyl, and limulus factor C are added to an endotoxin-comprising medium, and the emitted light is detected; or   (ii) the method is for testing pasteurization of dairy products the method comprising the steps of:
 a) providing a dairy product medium, 
 b) pasteurization of said dairy product medium, 
 c) adding said compound of the Formula I, wherein R 1  is phosphoryl, to the medium, optionally in combination with a buffer, so that the compound emits light, and 
 d) detecting the emitted light; or 
   (iii) the method is for testing of an antibiotic resistance in microorganisms, the method comprising the steps of:
 a) providing a medium comprising one or more microorganisms, 
 b) adding said compound of the Formula I, wherein R 1  is a beta-lactamase-labile group to the medium so that the compound emits light when antibiotic resistant microorganisms are present in the medium, and 
 c) detecting the emitted light; or 
   (iv) the method is for monitoring of a sterilization process, the method comprising the steps of:
 a) a1) providing a medium comprising a microorganism that produces alpha-glucosidase,
 a2) sterilizing the medium, 
 
 b) adding said compound of the Formula I, wherein R 1  is alpha-D-glucopyranosidyl, to the medium, and 
 c) detecting the emitted light; or 
   (v) the method is for detection of antibiotic resistance of bacteria and for antibiotic susceptibility testing, the method comprising the steps of:
 a) providing a medium comprising said bacteria, 
 b) adding an antibiotic, 
 c) adding said compound of the Formula I, wherein R 1  is an enzyme-labile group that is either removable or modified by an enzyme of said bacteria such that luminescence of said compound is triggered, wherein step c) may be performed before, together with, or after step b), and 
 d) detecting the emitted light, if any. 
   
     
     
         9 . The method of  claim 1 , wherein R 1  and the respective target analyte, target microorganism or target metabolite are defined as shown in the following table: 
       
         
           
                 
                 
               
                   R 1 
 
                   Target microorganism 
                 
                   acetyl 
                   a microorganism 
                 
                   butyryl 
                   a microorganism 
                 
                   octanoyl 
                   
                     Salmonella 
                   
                 
                   nonanoyl 
                   
                     Salmonella 
                   
                 
                   myo-inositol phosphoryl 
                   
                     Listeria monocytogenes; Bacillus; Staphylococcus; Clostridium; Mycobacterium tuberculosis 
                   
                 
                   phosphoryl 
                   
                     Staphylococcus aureus; Clostridium perfringens; S. agalactiae; Candida spp.; MRSA 
                   
                 
                   L-alaninyl 
                   Gram-negative bacteria; Yeast and molds 
                 
                   L-leucinyl 
                   Yeast and molds 
                 
                   β-alanyl 
                   
                     Pseudomonas aeruginosa 
                   
                 
                   L-pyroqlutamic acidyl 
                   
                     Enterococci; Streptococcus pyogenes; Citrobacter 
                   
                 
                   beta-D-qalactopyranosidyl 
                   Coliform;  E. coli 
 
                 
                   alpha-D-galactopyranosidyl 
                   
                     Salmonella 
                   
                 
                   alpha-D-glucopyranosidyl 
                   
                     Cronobacter sakazakii; 
                   
                 
                     
                     Staphylococcus aureus ; MRSA; VRE;  Geobacillus stearothermophilus   
                 
                   beta-D-glucopyranosidyl 
                   
                     Listeria spp ESBL producing enterobacteria Vibrio Enterococci VRE Candida spp. Clostridium difficile 
                   
                 
                   beta-D-qlucuronyl 
                   
                     E. coli; Streptococcus agalactiae 
                   
                 
                   beta-D-glucuronyl sodium salt 
                   
                     E. coli; Streptococcus agalactiae 
                   
                 
                   n-acetyl-beta-D-galactosaminidyl 
                   
                     Candida albicans 
                   
                 
                   N-acetylneuraminidyl 
                   
                     Prevotella 
                   
                 
                   cellobiosidyl 
                   
                     Cronobacter sakazakii 
                   
                 
                   ribofuranosidyl 
                   
                     Shigella 
                   
                 
                   choline phosphoryl 
                   
                     Bacillus 
                   
                 
                   -NO 2 
 
                   activity microorganism 
                 
                   Boc-Val-Pro-Arqininyl 
                   Bacterial Endotoxines 
                 
                   Boc-Asp(OBzl)-Pro-Arqininyl 
                   Bacterial Endotoxines 
                 
                   SucOMe-Arg-Pro-Tyrosinyl 
                   
                     Legionella pneumophila 
                   
                 
                   A beta-lactamase-labile group 
                   Antibiotic resistant microorganisms 
                 
                   Ac-QLQ-Ac-FQLQ-Ac-EFQLQ-Ac-DEFQLQ- 
                   a norovirus 
                 
                   Amides of 5-substituted-o-antranilic acid methyl ester 
                   
                     Campylobacter jejuni 
                   
                 
             
                
               
               
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       . 
     
     
         10 . The method of  claim 9 , wherein:
 (i) R 1  is acetyl, and said microorganism is  Campylobacter jejuni ,  C. coli  or  C. lari ;   (ii) R 1  is butyryl, and said microorganism is  Moraxella catarrhalis ; or   (iii) R 1  is a beta-lactamase-labile group and said beta-lactamase labile group is a beta-lactam antibiotic.   
     
     
         11 . The method of  claim 10 , wherein said beta-lactam antibiotic is a penicillin, a cephalosporin of generation 1 to 5, a cephamycin, or a carbapenem.