Patent Publication Number: US-6210911-B1

Title: Rapid Identification of Escherichia coli O157:H7

Description:
BACKGROUND OF THE INVENTION 
       Escherichia coli  (“ E. coli ”) O157:H7, first isolated in 1975 from a patient with grossly bloody diarrhea, is now recognized as an important foodborne pathogen (Ministry of Health and Welfare, Information on the Detection of Pathogenic Microorganisms, 1996; Meng et al., Trends Food Sci. Tech. 5:179-185, 1994; Padhye et al., J. Food Prot. 55:555-565, 1992; Riley et al., N. Engl. J. Med. 308:681-685, 1983). In adults, the illness is usually self-limited. However, the more serious hemolytic uremia syndrome (“HUS”) affects some of the infected patients, especially children and the elderly. The mortality rate of HUS is 3 to 10%. Most outbreaks have been associated with consumption of undercooked ground beef or raw milk. Cattle has been identified as an important reservoir of  E. coli  O157:H7. Person-to-person transmission has also been identified in some day-care center and nursing home outbreaks (Centers for Disease Control and Prevention, Morbid. Mortal. Weekly Rep. 42:253-257, 1993; Lior, Dairy Food and Environ. Sanitation 14:378-382, 1994; Padhye et al., J. Food Prot. 55:555-565, 1992). 
     Several methods have been developed for rapid detection and identification of  E. coli  O157:H7. Most of these methods are immunoassays for detecting the  E. coli  O157 somatic antigen (Huang et al., J. Food Prot. 59:170-174, 1996; Meng et al., Sci. Tech. 5:179-185, 1994). Confirmation of a positive result by either biochemical or serological tests, such as Vero cell assay or a test for the presence of H7 antigen, are required. 
     DNA probes and polymerase chain reaction have also been used to detect  E. coli  O157:H7 (Meng et al., Sci. Tech. 5:179-185, 1994). Although DNA-based methods are sensitive, there are several major drawbacks. First, they detect the presence of nucleic acid of the target bacteria rather than the viable bacteria themselves. Second, most of the DNA-based methods are designed to detect the genes that encode verotoxin or virulence-associated marker of verotoxin-producing  E. coli , and are not specific for  E. coli  O157:H7. In addition, DNA-based methods are cumbersome and expensive. 
     A coliphage named AR1 has been found to infect  E. coli  O157:H7 with high specificity (Ronner et al., Journal of Food Protection 54: 944-947, 1990). 
     SUMMARY OF THE INVENTION 
     The invention features a method of determining whether a test microorganism is a known microorganism such as a bacterium (e.g., an enterobacterium such as  Escherichia coli ), or yeast. The method includes the steps of: (i) providing a first culture that contains the test microorganism and an agent (e.g., a bacteriophage) that specifically affects the growth rate of the known microorganism; (ii) measuring a growth rate-related value of the first culture; and (iii) comparing the value of the first culture with a corresponding value of a second culture, the second culture being identical to the first culture except that the second culture is free of the agent, or contains the known microorganism and is free of the agent; wherein a difference in the two values is an indication that the test microorganism is the known microorganism. Examples of growth-rate-affecting agents include, but are not limited to, AR1 phage for  E. coli  O157:H7, P22 phage for  Salmonella typhimurium  (Griffiths, J. Dairy Sci., 76:3118-3125, 1993), and A511 phage for Listeria (Stewart et al., ASM News, 62:297-301, 1996). By “specifically” is meant that the agent affects the growth of mainly one microorganism. Of course, an agent that has cross-activity to a very limited number (e.g., no more than 3) of other microorganisms may also be used, if additional discerning criteria are available. 
     Also featured in the invention is a method of determining whether a test microorganism is  E. coli  O157:H7. 
     One embodiment of the method includes the following steps: (1) determining whether the test microorganism is  E. coli ; (2) growing the test microorganism in a medium containing sorbitol; (3) determining whether the test microorganism ferments sorbitol; (4) providing a first culture that contains the test microorganism and AR1 phage; (5) measuring a growth rate-related value of the first culture; and (6) comparing that value of the first culture with a corresponding value of a second (i.e., control) culture that is identical to the first culture except that (a) it is free of AR1 phage, or (b) it is free of AR1 phage and contains any  E. coli  strain instead of the test microorganism. The test microorganism is indicated as  E. coli  O157:H7 if (i) it is  E. coli , (ii) it is incapable of fermenting sorbitol, and (iii) there is a significant (e.g., at least two-fold) difference in the growth rate-related value between the two cultures. 
     In another embodiment of the method of this invention, the test microorganism is already known to be  E. coli . To determine if this  E. coli  strain is O157:H7, one can (1) grow the test strain in a sorbitol-containing medium; (2) determine whether the test strain ferments sorbitol; (3) provide a first culture that contains the test strain and AR1 phage; (4) measuring a growth rate-related value of the first culture; and (5) compare that value of the first culture with a corresponding value of a second culture which is identical to the first culture except that (a) it is free of AR1 phage, or (b) it contains any other  E. coli  strain and is free of AR1 phage. The test strain will be identified as  E. coli  O157:H7 if (i) it is incapable of fermenting sorbitol; and (ii) there is a significant (e.g., at least two-fold) difference in the growth rate-related value between the two cultures. 
     Yet another embodiment of the method is to determine whether a test microorganism incapable of fermenting sorbitol is  E. coli  O157:H7. This method includes the following steps: (1) determining whether the test microorganism is indicated as  E. coli ; (2) providing a first culture that contains the test microorganism and AR1 phage; (3) measuring a growth rate-related value of said first culture; and (4) comparing that value of the first culture with a corresponding value of a second culture that is identical to the first culture except that (a) it is free of AR1 phage, or (2) it is free of AR1 phage and contains any  E. coli  strain. The test microorganism is  E. coli  O157:H7 if (i) it is  E. coli ; and (ii) there is a significant (e.g., at least 10-fold, or even 20-fold) difference in the growth rate-related value between the two cultures. 
     If the test microorganism is already known to be susceptible to AR1 infection, one can conduct the following steps to determine if the microorganism is  E. coli  O157:H7: (1) determining whether the test microorganism is  E. coli ; (2) growing the test microorganism in a culture medium containing sorbitol; and (3) determining whether the test microorganism ferments sorbitol. The test microorganism is  E. coli  O157:H7 if it is  E. coli  and is incapable of fermenting sorbitol. 
     In all of the above-described methods, a growth rate-related value includes, but is not limited to, (i) a value of an electrical parameter such as conductance, resistance, or any other proper parameter; (ii) a value derived from values of an electrical parameter, e.g., a time point at each an accelerating change of an electrical parameter occurs (if the electrical parameter is conductance, the time point is herein termed “detection time”; (iii) a value of an optical parameter such as optical density; (iv) a value derived from values of an optical parameter; and (v) certain biochemical indexes that reflect growth of a microorganism. The methods based on use of an electrical or optical parameter allows automated screening of a large number of samples. 
    
    
     Other features and advantages of the present invention will be apparent from the following drawings and description, and also from the appending claims. 
     BRIEF DESCRIPTION OF THE DRAWING 
     FIG. 1 is a graph showing the effect of cell:phage ratio on the conductance curves of  E. coli  O157:H7 CCRC13095). Curve (a) was obtained in the absence of phage; and the remaining curves were obtained at the following cell:phage ratios: (b) 1:0.01, (c) 1:0.1, (d) 1:1, and (e) 1:10. 
     FIG. 2 is a graph showing the effect of cell:phage ratio on the coductance curves of non-O157:H7  E. coli  (CCRC15990). Curve (a) was obtained in the absence of phage; and the remaining curves were obtained at the following cell:phage ratios: (b) 1:1, (c) 1:10, and (d) 1:100. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention features methods of determining whether a test microorganism is  E. coli  O157:H7. The invention is based on Applicants&#39; discovery that a test microorganism can be identified as  E. coli  O157:H7 with high (e.g., over 90% or even 98%) certainty if it meets the following criteria: The microorganism must be (1)  E. coli ; (2) incapable of fermenting sorbitol; and (3) susceptible to infection by AR1 phage (e.g., as determined by a conductance-based protocol described below). Various strains of AR1 phage can be used to practice methods of this invention. A strain of AR1 phage was deposited on Dec. 1, 1994 at the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, U.S.A. The ATCC accession number is 75957. 
     There are several widely known assays for determining whether a microorganism is  E. coli  (Venkateswaran et al., Applied and Environmental Microbiology, 62: 2236-2243, 1996; Hitchins et al., Chapter 4, in FDA Bacteriological Analytical Manual, 1995; and Okrend et al., Revision 3 of Laboratory Communication #38, pages 1-13, FSIS, Microbiology Division, U.S. Department of Agriculture, Washington, D.C., 1989); conventional biochemical tests or a commercial kit such as API 20E (bioMerieux, Marcy-I&#39;Etoile, France) can be used. 
     Assays for determining whether a microorganism isolate can ferment sorbitol are well known in the art. In a typical assay, test microorganisms or a sample suspected of containing bacteria (e.g., food, blood, stool, or drinking water) are cultured on MacConkey sorbitol agar to form colonies. If a colony can ferment sorbitol, it will be red; otherwise, it will be white. 
     To determine whether a microorganism isolate is susceptible to infection by AR1 phage, plaque assay on agar plates can be performed (see, e.g., Ronner et al., supra). Alternatively, the isolate can be used to inoculate an appropriate liquid culture medium together with an appropriate amount of AR1 phage (see the Example, infra). If the microorganism is infectable by AR1 phage, the liquid culture will not reach an exponential growth phase (i.e., log phase), which is characteristic of a healthy culture; or the culture will grow much slower and reach the log phase at a significantly later time (i.e., at least twice or even thrice as long) than a control culture that is free of AR1 phage and is inoculated with an identical or similar (i.e., the difference is no more than 10-fold) concentration of an  E. coli  strain or the microorganism isolate. Thus, an artisan can compare the time for each of the test and control cultures to reach the log phase. If the control culture contains a known  E. coli  strain, the time for it to reach the log phase can be predetermined or estimated from prior art. Alternatively, one can compare a growth rate-related value (such as an electrical or an optical parameter) of the culture with that of the control culture (which can be pre-determined or estimated from prior art) at a given time point. The time point can be empirically pre-determined. If the test culture is infected by AR1 phage, there will be a significant difference in the growth rate-related value between the test and control cultures. 
     For the purpose of identifying  E. coli  O157:H7, it is preferred to use an electrical parameter (e.g., conductance)-based assay, rather than a plaque assay, to determine a test microorganism&#39;s susceptibility to AR1. The former assay not only gives a lower background, but also is easier to be adapted to automation. 
     The three criteria for identifying  E. coli  O157:H7 can be examined in any order. For instance, one can first grow a test microorganism on MacConkey sorbitol agar to determine if the microorganism is a sorbitol fermenter; if not, one can proceed to further determine if the microorganism is infectable by AR1 phage; if yes, then one can determine if the test microorganism is  E. coli . Alternatively, one can first determine if the test microorganism is infectable by AR1 phage, then determine if it can ferment sorbitol and last if it is  E. coli . The three criteria can also be examined concurrently. Or if one or two of the criteria is already known to be met, an artisan can simply test for the remaining criteria or criterion. 
     Without further elaboration, it is believed that the above description has enabled the present invention to its fullest extent. All citations are incorporated herein by reference. The following specific example is, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. 
     Identification of  E. coli  O157:H7 Based on a Conductance 
     Conductance measurements have been used for the determination of total bacterial count Enterobacteriaceae bacteria and other bacteria in food samples (Firstenberg-Eden, R. Food Tech. 37:64-70, 1983; Gibson et al., Int. J. Food Microbiol. 1:127-134, 1984; Hadley et al., Appl. Environ. Microbiol. 34:14-17, 1977; Cousins et al., J. Food Prot. 53:568-570, 1990; Easter et al., J. Hyg. Camb. 94:245-262, 1985; Smith et al., J. Appl. Bacteriol. 67:575-588, 1989). They have also been used for detecting the presence of antibiotics in bacterial cultures (U.S. Pat. No. 5,591,599 to Chang et al.). The principle of these measurements is that multiplication of bacteria in a culture medium will result in an increase in the conductivity of the medium. At a time point (“detection time”) when the bacteria concentration reaches approximately 10 7  cfu/ml, an accelerating increase of conductivity will occur. Detection times are inversely proportional to the initial bacterial count in the test sample (Firstenberg-Eden et al., Impedance Microbiology. pp. 7-90. John Wiley and Sons Inc., New York, 1984). 
     The technique is particularly useful for rapid screening various foods to determine whether they meet the desired microbiological standards. However, this technique is for the first time used to identify  E. coli  O157:H7 by using AR1 phage which is specific to the microorganism. 
     Bacterial Strains and Media 
     All the bacterial strains used herein are listed in Table 1. There were 41 strains of  E. coli  O157:H7 and 155 strains of non-O157:H7  E. coli . Among the non-O157:H7  E. coli  strains, which include food isolates, enteroinvasive  E. coli  (“EIEC” ), enteropathogenic  E. coli  (“EPEC” ), and enterotoxigenic  E. coli  (“ETEC” ), the serotypes of 99 strains were known; and the remaining 56 strains were only tested for O157 antigen by a latex test (Oxoid, Unipath Ltd., Hampshire, UK) and found to be negative. 
     All the bacterial strains were maintained at 4° C. on tryptic soy agar (Difco Laboratories, Detroit, Mich, USA) except that strains of  Vibrio parahaemolyticus  were maintained on tryptic soy agar supplemented with 2.5% NaCl. 
     The following media were used: MacConkey sorbitol agar (Oxoid, Unipath Ltd., Hampshire, UK), 0.1% peptone water, Plate Count Agar (Difco Laboratories, Detroit, Mich.), and Malthus SPYE broth (Malthus Instruments, Crawley, UK). 
     
       
         
           
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Microorganism 
                 No. of Strains 
                 Source a   
               
               
                   
               
             
            
               
                   Escherichia coli  O157:H7 
                  41 
                 CRC 13084-99, 
               
               
                   
                   
                 14824-5; 15373-4, 
               
               
                   
                   
                 15376-7, 15970; 
               
               
                   
                   
                 H11-18, NCHU; W1, 
               
               
                   
                   
                 NLFD; LA1, LO3-8, 
               
               
                   
                   
                 A8993-C32, 933, 
               
               
                   
                   
                 NTU 
               
               
                   E. coli  O113:K75(B19):H21 
                 1 
                 CCRC 14883 
               
               
                   E. coli  O121:H12 
                 1 
                 CCRC 14884 
               
               
                   E. coli  O145:K?(B):H- 
                 1 
                 CCRC 14885 
               
               
                   E. coli  O8:K27-:H- 
                 1 
                 CCRC 14909 
               
               
                   E. coli  O26 
                 1 
                 CCRC 14917 
               
               
                   E. coli  O111 
                 1 
                 CCRC 14918 
               
               
                   E. coli  O25:K98:NM 
                 1 
                 CCRC 15370 
               
               
                   E. coli  O78:K80:H12 
                 1 
                 CCRC 15371 
               
               
                   E. coli  O124:NM 
                 1 
                 CCRC 15375 
               
               
                   E. coli  O1a,1b: 
                 1 
                 CCRC 15479 
               
               
                   E. coli  O2a,2b:K5(L):H4 
                 1 
                 CCRC 15480 
               
               
                   E. coli  O3:K2a,2b(L):H2 
                 1 
                 CCRC 15481 
               
               
                   E. coli  O5:K4(L):H4 
                 1 
                 CCRC 15482 
               
               
                   E. coli  O6:K2:H1 
                 1 
                 CCRC 15483 
               
               
                   E. coli  O8:K85:K99 
                 1 
                 CCRC 15484 
               
               
                   E. coli  O9:K35:K99 
                 1 
                 CCRC 15485 
               
               
                   E. coli  O11:H4 
                 1 
                 CCRC 15486 
               
               
                   E. coli  O13:K(L):H11 
                 1 
                 CCRC 15487 
               
               
                   E. coli  O14:K(7L):NM 
                 1 
                 CCRC 15488 
               
               
                   E. coli  O15:K14(L):H4 
                 1 
                 CCRC 15489 
               
               
                   E. coli  O16:K1(L):NM 
                 1 
                 CCRC 15490 
               
               
                   E. coli  O17:H16(L):H18 
                 1 
                 CCRC 15491 
               
               
                   E. coli  O19a,19b:K:H7 
                 1 
                 CCRC 15492 
               
               
                 
                   E. coli 
                 
                 1 
                 CCRC 15493 
               
               
                 O20a,20b:K17(L):NM 
               
               
                   E. coli  O22:K13(L):H1 
                 1 
                 CCRC 15494 
               
               
                   E. coli  O26:K60(B6) 
                 1 
                 CCRC 15496 
               
               
                 
                   E. coli 
                 
                 1 
                 CCRC 15497 
               
               
                 O28a,28c:K73(B18):NM 
               
               
                   E. coli  O29:NM 
                 1 
                 CCRC 15498 
               
               
                   E. coli  O32:K:H19 
                 1 
                 CCRC 15499 
               
               
                   E. coli  O34:K:H10 
                 1 
                 CCRC 15500 
               
               
                   E. coli  O35:K:H10 
                 1 
                 CCRC 15501 
               
               
                   E. coli  O37:K:H10 
                 1 
                 CCRC 15502 
               
               
                   E. coli  O88:K:H25 
                 1 
                 CCRC 15510 
               
               
                   E. coli  O101:K30:K99 
                 1 
                 CCRC 15511 
               
               
                   E. coli  O107:K:H27 
                 1 
                 CCRC 15512 
               
               
                   E. coli  O114:K:H32 
                 1 
                 CCRC 15513 
               
               
                   E. coli  O119:K69(B14) 
                 1 
                 CCRC 15514 
               
               
                   E. coli  O124:K72(B17):H 
                 1 
                 CCRC 15515 
               
               
                   E. coli  O139:K82(B):H1 
                 1 
                 CCRC 15518 
               
               
                   E. coli  O36:K:H9 
                 1 
                 CCRC 15519 
               
               
                   E. coli  O41:K:H40 
                 1 
                 CCRC 15520 
               
               
                   E. coli  O55:B5:H- 
                 1 
                 CCRC 15521 
               
               
                   E. coli  O60:K:H33 
                 1 
                 CCRC 15522 
               
               
                   E. coli  O65:K:NM 
                 1 
                 CCRC 15523 
               
               
                   E. coli  O70:K:H42 
                 1 
                 CCRC 15524 
               
               
                   E. coli  O77:K:NM 
                 1 
                 CCRC 15526 
               
               
                   E. coli  O86a,86b:K64(B9) 
                 1 
                 CCRC 15527 
               
               
                   E. coli  O103:K:H8 
                 1 
                 CCRC 15529 
               
               
                 
                   E. coli 
                 
                 1 
                 CCRC 15530 
               
               
                 O111a,111b:K58:H21 
               
               
                   E. coli  O116:K:H10 
                 1 
                 CCRC 15531 
               
               
                   E. coli  O120:K:H6 
                 1 
                 CCRC 15532 
               
               
                 
                   E. coli 
                 
                 1 
                 CCRC 15533 
               
               
                 O125a,125c:K70(B15):H 
               
               
                 
                   E. coli 
                 
                 1 
                 CCRC 15534 
               
               
                 O128a,128b:K67(B12):H 
               
               
                   E. coli  O138:K81(B):H14 
                 1 
                 CCRC 15535 
               
               
                   E. coli  O142:K86(B):H6 
                 1 
                 CCRC 15536 
               
               
                 
                   E. coli 
                 
                 1 
                 CCRC 15869 
               
               
                 Ol8a,18c:K77(B21):H7 
               
               
                   E. coli  O38:K:NM 
                 1 
                 CCRC 15870 
               
               
                   E. coli  O81:K:NM 
                 1 
                 CCRC 15872 
               
               
                   E. coli  O83:K:H31 
                 1 
                 CCRC 15873 
               
               
                   E. coli  O86:K61(B7) 
                 1 
                 CCRC 15874 
               
               
                   E. coli  O86a:K61 
                 1 
                 CCRC 15875 
               
               
                   E. coli  O91:K:NM 
                 1 
                 CCRC 15876 
               
               
                   E. coli  O101:K-:K99 
                 1 
                 CCRC 15877 
               
               
                   E. coli  O10:K5(L):H4 
                 1 
                 CCRC 15878 
               
               
                 
                   E. coli 
                 
                 1 
                 CCRC 15879 
               
               
                 O112a,112b:K68(B13) 
               
               
                 
                   E. coli 
                 
                 1 
                 CCRC 15880 
               
               
                 O125a,125b:K70(B15):H 
               
               
                 
                   E. coli 
                 
                 1 
                 CCRC 15881 
               
               
                 O128a,128c:K67(B12):H 
               
               
                   E. coli  O129:(K):H11 
                 1 
                 CCRC 15882 
               
               
                   E. coli  O4 
                 1 
                 CCRC 15924 
               
               
                   E. coli  O7:K1(L):NM 
                 1 
                 CCRC 15925 
               
               
                   E. coli  O8:K8(L):H4 
                 1 
                 CCRC 15926 
               
               
                   E. coli  O9:K9(B):H12 
                 1 
                 CCRC 15927 
               
               
                   E. coli  O11:K10(L):H10 
                 1 
                 CCRC 15928 
               
               
                   E. coli  O12:K5(L):NM 
                 1 
                 CCRC 15929 
               
               
                   E. coli  O15 
                 2 
                 CCRC 15930-1 
               
               
                   E. coli  O16:K92:H- 
                 1 
                 CCRC 15932 
               
               
                   E. coli  O18 
                 1 
                 CCRC 15933 
               
               
                   E. coli  O21:K20(L):NM 
                 1 
                 CCRC 15934 
               
               
                   E. coli  O24 
                 1 
                 CCRC 15935 
               
               
                   E. coli  O25 
                 1 
                 CCRC 15936 
               
               
                   E. coli  O27:K:NM 
                 1 
                 CCRC 15937 
               
               
                   E. coli  O28 
                 1 
                 CCRC 15938 
               
               
                   E. coli  O38:K:H26 
                 1 
                 CCRC 15939 
               
               
                   E. coli  O44 
                 1 
                 CCRC 15940 
               
               
                   E. coli  O48 
                 1 
                 CCRC 15941 
               
               
                   E. coli  O73 
                 1 
                 CCRC 15942 
               
               
                   E. coli  O75 
                 1 
                 CCRC 15943 
               
               
                   E. coli  O87 
                 1 
                 CCRC 15944 
               
               
                   E. coli  O90 
                 1 
                 CCRC 15945 
               
               
                   E. coli  O102 
                 1 
                 CCRC 15946 
               
               
                   E. coli  O111 
                 1 
                 CCRC 15947 
               
               
                 
                   E. coli 
                 
                 1 
                 CCRC 15948 
               
               
                 O112a,112c:K66(B11):NM 
               
               
                   E. coli  O126:K71(B16):H 
                 1 
                 CCRC 15949 
               
               
                   E. coli  O135:K:NM 
                 1 
                 CCRC 15950 
               
               
                   E. coli  O139 
                 1 
                 CCRC 15951 
               
               
                   E. coli  O2:K7(56)1(B1):H7 
                 1 
                 CCRC 15969 
               
               
                   E. coli  O153:K-:H7 
                 1 
                 CCRC 15990 
               
               
                 
                   E. coli 
                 
                 1 
                 CCRC 15991 
               
               
                 O157:K88(:F4)ac:H19 
               
               
                 
                   E. coli 
                 
                 8 
                 NCHU 
               
               
                 (enterotoxigenic, serotype 
               
               
                 unknown) 
               
               
                 
                   E. coli 
                 
                 2 
                 NCHU 
               
               
                 (enteroinvasive, serotype 
               
               
                 unknown) 
               
               
                 
                   E. coli 
                 
                 1 
                 NCHU 
               
               
                 (enteropathogenic, 
               
               
                 serotype unknown) 
               
               
                   E. coli  (food isolates, 
                  45 
                 CCRC 
               
               
                 serotype unknown) 
               
               
                 
                   E. blattae 
                 
                 1 
                 CCRC 15589 
               
               
                 
                   E. fergusonii 
                 
                 5 
                 CCRC 15582-6 
               
               
                 
                   E. hermannii 
                 
                 2 
                 CCRC 15587-8 
               
               
                 
                   E. vulneris 
                 
                 5 
                 CCRC 15952-6 
               
               
                 
                   Aeromonas schubertii 
                 
                 1 
                 CCRC 14138 
               
               
                 
                   Citrobacter freundii 
                 
                 3 
                 CCRC 10637, 12291-2 
               
               
                 
                   Enterobacter aerogenes 
                 
                 1 
                 CCRC 10370 
               
               
                 
                   Enterobacter cloacae 
                 
                 1 
                 CCRC 12313 
               
               
                 
                   Enterococcus durans 
                 
                 1 
                 CCRC 10790 
               
               
                 
                   Enterococcus faecalis 
                 
                 1 
                 CCRC 10789 
               
               
                 
                   Enterococcus faecium 
                 
                 3 
                 CCRC 10067, 12808-9 
               
               
                   Erwinia ananas  pv.  ananas   
                 1 
                 CCRC 12150 
               
               
                 
                   Erwinia chrysanthemi 
                 
                 1 
                 CCRC 10317 
               
               
                 
                   Klebsiella oxytoca 
                 
                 1 
                 CCRC 10026 
               
               
                 
                   Klebsiella pneumoniae 
                 
                 5 
                 CCRC 10693-4, 
               
               
                 subsup.  pneumoniae   
                   
                 11546, 11644, 
               
               
                   
                   
                 12284 
               
               
                 
                   Morganella morganii 
                 
                 1 
                 CCRC 11257 
               
               
                 
                   Proteus mirablis 
                 
                 2 
                 CCRC 10725, 10727 
               
               
                 
                   Proteus penneri 
                 
                 1 
                 CCRC 14123 
               
               
                 
                   Proteus myxofaciens 
                 
                 1 
                 CCRC 12222 
               
               
                 
                   Proteus vulgaris 
                 
                 3 
                 CCRC 110486, 
               
               
                   
                   
                 10728, 14882 
               
               
                 
                   Pseudomonas aeruginosa 
                 
                 1 
                 CCRC 11633 
               
               
                 
                   Pseudomonas aureofaciens 
                 
                 1 
                 CCRC 11057 
               
               
                 
                   Pseudomonas mendocina 
                 
                 1 
                 CCRC 10458 
               
               
                 
                   Pseudomonas vesicularis 
                 
                 1 
                 CCRC 11012 
               
               
                 
                   Salmonella arizonae 
                 
                 1 
                 CCRC 10742 
               
               
                 
                   Salmonella dublin 
                 
                 1 
                 CCRC 13852 
               
               
                 
                   Salmonella enteritidis 
                 
                 1 
                 CCRC 10744 
               
               
                   Salmonella paratyphi  A 
                 1 
                 CCRC 14878 
               
               
                   Salmonella paratyphi  B 
                 1 
                 CCRC 14879 
               
               
                 
                   Salmonella typhi 
                 
                 1 
                 CCRC 12948 
               
               
                 
                   Salmonella typhimurium 
                 
                 1 
                 CCRC 12459 
               
               
                 
                   Serratia grimesii 
                 
                 1 
                 CCRC 10767 
               
               
                 
                   Serratia odorifera 
                 
                 1 
                 CCRC 12223 
               
               
                 
                   Serratia plymuthica 
                 
                 1 
                 CCRC 12224 
               
               
                 
                   Shigella boydii 
                 
                 6 
                 CCRC 10771, 15957-61 
               
               
                 
                   Shigella dysenteria 
                 
                 1 
                 CCRC 13983 
               
               
                 
                   Shigella flexneri 
                 
                 3 
                 CCRC 10772, 13984, 
               
               
                   
                   
                 15962 
               
               
                 
                   Shigella sonnei 
                 
                 3 
                 CCRC 10773-4, 
               
               
                   
                   
                 15966 
               
               
                 
                   Vibrio parahaemolyticus 
                 
                 5 
                 CCRC 12863-4, 
               
               
                   
                   
                 13023, 13025, 
               
               
                   
                   
                 13027 
               
               
                 
                   Yersubua enterocolitica 
                 
                 1 
                 CCRC 139999 
               
               
                   
               
               
                   a ATCC, American Type Culture Collection, Rockville, Maryland, U.S.A.  
               
               
                 CCRC, Culture Collection and Research Center, Hsinchu, Taiwan, R.O.C.  
               
               
                 NCHU, National Chung-hsing University, Taichung, Taiwan, R.O.C.  
               
               
                 NLFD, National Laboratories of Food and Drugs, Taipei, Taiwan, R.O.C.  
               
               
                 NTU, National Taiwan University, Taiwan, R.O.C.  
               
            
           
         
       
     
     Procedures of the Conductance Method 
     Bacterial strains were grown on MacConkey sorbitol agar medium at 35° C. for 18 to 24 hrs. The ability of each strain to ferment sorbitol was recorded: colonies of a strain capable of fermenting sorbitol are red, whereas colonies of a strain incapable of fermenting sorbitol are white. A single colony of each strain was suspended in 10 ml of 0.1% peptone water (approximately 10 7  cfu/ml) and serially diluted 1:10 with the same peptone water. AR1 phage was prepared with a method used for λ phage isolation (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and serially diluted 1:10 in a phage buffer (Ronner et al., J. Food Prot. 53:944-947, 1990). Titer of the phage was determined with a plaque assay (Snustad et al., Genetics Experiments With Bacterial Viruses, W. H. Freeman and Company, San Francisco, Calif., 1971). Conductance measurement was performed with the Malthus 2000 microbiological analyzer (Malthus Instruments, Crawley, UK). The conductivity cell was a 10-ml tube fitted with platinum electrodes printed on a ceramic base. Each tube containing 5 ml of SPYE broth was inoculated with 0.5 ml of the bacterial suspension and 0.5 ml of the phage suspension. The tubes were incubated in the Malthus analyzer at 35° C., and conductance change in each tube was automatically scanned at 6-min intervals for 22 h. Readouts were available in numerical or graphic form. The analyzer automatically determined in each tube the time point when an accelerating increase in conductance occurred, i.e., when the conductance increased 1 μS or more for three consecutive readings. This time point is termed “detection time.” One hundred and twenty samples can be analyzed simultaneously by the instrument. 
     Definition of Positives, Negatives, Test Sensitivity, and Specificity 
     An  E. coli  isolate having white colonies on MacConkey sorbitol agar and having no detection time within 22 h was considered  E. coli  O157:H7 and was defined as a positive. An  E. coli  isolate having red colonies on McConkey sorbitol agar and having detection time within 22 h was not considered  E. coli  O157:H7 and was defined as a negative. The sensitivity of the conductance method was defined as the percentage of  E. coli  O157:H7 strains that were found to be a positive by the method. The specificity was defined as the percentage of non-O157:H7  E. coli  strains that were found to be a negative (McClure, J. Assoc. Off. Anal. Chem. 73:953-960, 1990). 
     Optimization of the Conductance Method 
     Operation parameters (e.g., inoculation concentration of bacterial cells, and cell to phage ratio) were evaluated prior to a large scale of test. In the absence of AR1 phage, both  E. coli  CCRC 13095 (an O157:H7 strain) and  E. coli  CCRC 15990 (a non-O157:H7 strain) displayed similar patterns of conductivity change during the course of culturing. Further, for both strains, detection times were inversely proportional to the inoculation levels. At inoculation concentrations between 10 6  and 10 7  cfu/ml, the detection times were less than 3 h. 
     When AR1 phage was added to the medium inoculated with  E. coli , however, great difference was observed in detection times between O157:H7  E. coli  and non-O157:H7  E. coli  strains. Table 2 shows the effect of various  E. coli  inoculation concentrations and cell to phage ratios on the detection time. When the inoculation concentration was between 10 6  and 10 7  cfu/ml, there was a significant increase in the detection time of  E. coli  O157:H7 (CCRC 13095) at a cell:phage ratio of 1:0.1 as compared to a cell:phage ratio of 1:0.01 or higher. At a cell:phage ratio of 1:10, no detection time was obtained over an incubation period of 22 h, indicating that growth of the bacteria was completely inhibited. In contrast, the detection times of non-O157:H7  E. coli  remained unchanged even at a cell:phage ratio of 1:100. FIGS. 1 and 2 illustrate the changes of the conductance curves (or the detection times) of  E. coli  CCRC 13095 (O157:H7) and of  E. coli  CCRC 15990 (non-O157:H7) in the presence of AR1 phage. Specifically, the detection time of  E. coli  CCRC 13095 was 1.7 h in the absence of AR1 phage, and was 1.7, 16.4, &gt;24, and &gt;24 h when the cell:phage ratio was 1:0.01, 1:0.1, 1:1, and 1:10, respectively. The detection time of  E. coli  CCRC 15990, on the other hand, was 2.6 h in the absence of phage, and was 2.6, 2.6 and 2.7 h when the cell:phage ratio was 1:1, 1:10, and 1:100, respectively. 
     An inoculation concentration of 10 7  cfu/ml (which can be achieved by suspending a single colony in approximately 10 ml of 0.1% peptone water) was used for the test of other bacteria. In addition, to achieve a complete growth inhibition for  E. coli  O157:H7, a cell:phage ratio of 1:10 was used for further studies. Under these conditions, detection times of non- E. coli  O157:H7 bacteria would be obtained within a few hours, while no detection time would be obtained within 22 h for  E. coli  O157:H7 strains. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Effect of cell:phage ratio on the detection times 
               
               
                 of  E. coli  O157:H7 (CCRC 13095) and of non-O157:H7  E. coli   
               
               
                 (CCRC 15990) a   
               
            
           
           
               
               
               
            
               
                   
                 Cell:phage ratio 
                 Detection time (h) 
               
            
           
           
               
               
            
               
                   
                 Inoculation concentration of bacterium (cfu/ml) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Microorganism 
                   
                 10 4   
                 10 5   
                 10 6   
                 10 7   
               
               
                   
               
               
                 
                   E. coli 
                 
                 — b   
                 4.7 ± 0.1 
                 3.6 ± 0.3 
                 2.6 ± 0.3 
                 ND c   
               
               
                 CCRC 15990 (non- 
                 1:1 
                 5.0 ± 0.1 
                 3.6 ± 0.2 
                 2.6 ± 0.1 
                 ND c   
               
               
                 O157:H7) 
                 1:10 
                 4.8 ± 0.1 
                 3.5 ± 0.1 
                 2.7 ± 0.1 
                 ND c   
               
               
                   
                 1:100 
                 4.7 ± 0.1 
                 3.8 ± 0.2 
                 2.8 ± 0.1 
                 ND c   
               
               
                 
                   E. coli 
                 
                 — b   
                 5.8 ± 0.5 
                 4.5 ± 0.1 
                 2.7 ± 0.3 
                 1.7 ± 0.2 
               
               
                 CCRC 13095 
                 1:0.01 
                 5.9 ± 0.2 
                 4.1 ± 0.1 
                 3.0 ± 0.2 
                 1.7 ± 0.2 
               
               
                 (O157:H7) 
                 1:0.1 
                 5.7 ± 0.2 
                 4.3 ± 0.2 
                 14.5 ± 0.1  
                 16.4 ± 3.0  
               
               
                   
                 1:1 
                 6.0 ± 0.3 
                 — d   
                 — d   
                 — d   
               
               
                   
                 1:10 
                 — d   
                 — d   
                 — d   
                 — d   
               
               
                   
                 1:100 
                 — d   
                 — d   
                 — d   
                 — d   
               
               
                   
               
               
                   a Mean of triplicate ± standard deviation  
               
               
                   b No phage was added to the conductance tube.  
               
               
                   c Not determined.  
               
               
                   d No detection time.  
               
            
           
         
       
     
     Identification of  E. coli  O157:H7 
     The optimal conditions for the conductance method as described above were applied to test a total of 269 bacterial strains (Table 1). Among the 269 strains, 41 were  E. coli  O157:H7; 99 were  E. coli  having serotypes other than O157:H7; 56 were non-O157 isolates of  E. coli  with serotypes undetermined; and 73 were other bacteria. 
     All 41 strains of  E. coli  O157:H7 were correctly identified: No detection times could be obtained for these strains during a 22 h incubation period (Table 3). Thus, the sensitivity of the test was 100% (41/41). Surprisingly, among the 155 non-O157:H7  E. coli  strains, 13 were found to be positive by the conductance method, and of all these 13 false-positives, 12 were sorbitol-fermenters (i.e., red colonies on MacConkey-sorbitol agar; Table 3) with the remaining  E. coli  0125a,125c:K70(B15):H being a sorbitol non-fermenter. In other words, a combination of the conductance test and the sorbitol fermentation test leads to a specificity as high as 99.4% (154/155) for determining if an  E. coli  isolate is O157:H7. 
     Four strains of Shigella spp., including one  S. boydii  (serotype 5) and two  S. flexneri  strains, produced false-positive results among the 73 strains of non- E. coli  bacteria (Table 3). However, these bacteria were not  E. coli , and hence there is no need to consider if they are  E. coli  O157:H7. 
     The main advantages of the conductance method are high sensitivity and specificity, low cost, and adaptability to automation. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Colony color of bacteria grown on MacConkey sorbitol agar and 
               
               
                 the results of conductance test. 
               
            
           
           
               
               
               
               
            
               
                   
                 Colony color on 
                   
                   
               
               
                   
                 MacConkey 
                   
                 Result of conductance test b   
               
            
           
           
               
               
               
               
               
            
               
                 Microorganism 
                 sorbitol agar a   
                 No. of strain 
                 + 
                 − 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   E. coli  O157:H7 
                 W 
                 41 
                 41 
                 0 
               
               
                 non-O157:H7  E. coli   
                 R 
                 87 
                 5 
                 82 
               
               
                 (serotypes determined) 
                 W 
                 12 
                 1 
                 11 
               
               
                 non-O157:H7  E. coli   
                 R 
                 50 
                 7 
                 43 
               
               
                 (serotypes unknown) 
                 W 
                 6 
                 0 
                 6 
               
               
                 
                   Escherichia blattae 
                 
                 W 
                 1 
                 0 
                 1 
               
               
                 
                   Escherichia fergunsonii 
                 
                 W 
                 5 
                 0 
                 5 
               
               
                 
                   Escherichia hermannii 
                 
                 W 
                 2 
                 0 
                 2 
               
               
                 
                   Escherichia vulneris 
                 
                 W 
                 5 
                 0 
                 5 
               
               
                 
                   Aeromonas schubertii 
                 
                 No growth 
                 1 
                 0 c   
                 0 c   
               
               
                 
                   Citrobacter freundii 
                 
                 R 
                 3 
                 0 
                 3 
               
               
                 Enterobacter spp. 
                 R 
                 2 
                 0 
                 2 
               
               
                 Enterococcus spp. 
                 No growth 
                 5 
                 0 c   
                 0 c   
               
               
                 Erwinia spp. 
                 W 
                 1 
                 0 
                 1 
               
               
                   
                 R 
                 1 
                 0 
                 1 
               
               
                 Klebsiella spp. 
                 R 
                 6 
                 0 
                 6 
               
               
                 
                   Morganella morganii 
                 
                 W 
                 1 
                 0 
                 1 
               
               
                 Proteus spp. 
                 W 
                 5 
                 0 
                 5 
               
               
                 Proteus spp. 
                 No growth 
                 2 
                 0 c   
                 0 c   
               
               
                 Pseudomonas spp. 
                 W 
                 3 
                 0 
                 3 
               
               
                   
                 No growth 
                 1 
                 0 c   
                 0 c   
               
               
                 Salmonella spp. 
                 R 
                 7 
                 0 
                 7 
               
               
                 Serratia spp. 
                 R 
                 2 
                 0 
                 2 
               
               
                   
                 No growth 
                 1 
                 0 c   
                 0 c   
               
               
                 Shigella spp. 
                 R 
                 3 
                 0 
                 3 
               
               
                   
                 W 
                 8 
                 3 
                 3 
               
               
                   
                 No growth 
                 2 
                 0 c   
                 0 c   
               
               
                 
                   Vibrio 
                 
                 W 
                 5 
                 0 
                 0 
               
               
                 
                   parahaemolyticus 
                 
                 W 
                 5 c   
                 0 c   
                 0 c   
               
               
                 
                   Yersinia enterocolitica 
                 
                 R 
                 1 
                 0 
                 1 
               
               
                   
               
               
                   a Sorbitol-fermenters have red (R) colonies and sorbitol non-fermenters have white (W) colonies on MacConkey sorbitol agar.  
               
               
                   b“+”  strain having no detection time within 22 h, “−” a strain having detection time within 22 h.  
               
               
                   c Test strains failed to grow either on MacConkey sorbitol agar or in SPYE broth.  
               
            
           
         
       
     
     Other Embodiments 
     From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims. 
     For instance, any phage that displays host specificity similar to AR1 can be used in lieu of AR1 in the present methods. Also, any analog of sorbitol and any biochemical process analogous to sorbitol fermentation can be used in lieu of sorbitol and sorbitol fermentation, respectively.